Different Transcutaneous Auricular Vagus Nerve Stimulation Parameters Modulate the Anti-Inflammatory Effects on Lipopolysaccharide-Induced Acute Inflammation in Mice

Figure 1. Experimental design and procedure of taVNS in the LPS-induced endotoxemia in mice. (A) Mice were bilaterally treated with taVNS, followed by the cymba and cavum concha of the vagus innervation area in the ear. (B) Under anesthesia, mice were stimulated with taVNS treatments for 5 or 10 min before and after LPS injection. The animals were euthanized after 2 h of LPS administration, and the whole blood and tissues were collected. (C) Four different taVNS parameters were used during stimulation in this study. (D) Schematic image showing the charge-balanced stimulation waveform of taVNS with low pulse frequency (top) and high pulse frequency (bottom).

Figure 2. A decrease in pro-inflammatory cytokine releases by taVNS in LPS-induced endotoxemia. (A) Mice were treated with taVNS and/or MLA treatment for acute inflammation from LPS. The expression levels of serum TNF-α and IL-1β were measured by ELISA. The decrease and recovery percentages of each cytokine were represented as a number. (B) The expression levels of serum TNF-α and IL-1β were determined using ELISA pre- and post-treatment of taVNS with different pulse frequency and time parameters. Data have been presented as the means and SD; * p < 0.05, ** p < 0.01, and *** p < 0.001, compared to the corresponding control.

Figure 3. taVNS reduced inflammation of the spleen in LPS-induced endotoxemia. (A) Representative images of the spleen after treatment of taVNS with different pulse frequencies and time parameters. Scale bar: 5 mm. (B) Western blot analysis using an antibody against MPO was used to analyze the relative expression levels of neutrophils in the indicated stimulation conditions of taVNS on LPS-induced inflammation. The intensity ratios for MPO were presented as a graph using ImageJ. (C) The mRNA levels of pro- and anti-inflammatory cytokines were determined by qPCR. All results have been presented as the means and SD; * p < 0.05, ** p < 0.01, and *** p < 0.001, compared to the control.

Figure 4. Anti-inflammatory effect of taVNS on the lung of LPS-induced endotoxemia. (A) The representative images of the lung after treatment of taVNS with 15 Hz and 25 Hz. Scale bar: 5 mm. The results of H&E staining were determined using the lung injury score upon observation under a light microscope. Scale bar: 10 μm. (B) MPO staining was determined by the infiltrated immune cells on the lung. Scale bar: 20 μm. (C) The relative scoring of lung injuries was compared as a graph in three independent images. Positively stained area was measured in three different images in each group and then represented on a graph. (D) The expression levels of pro- and anti-inflammatory cytokines genes were determined by qPCR, and significance was compared among groups. Data have been presented as the means and standard deviation (n = 5–10); * p < 0.05, ** p < 0.01, and *** p < 0.001, as compared to the corresponding control.

Figure 5. taVNS induced anti-inflammatory effect on the intestine of LPS-induced endotoxemia. (A) Determination of the pro-inflammatory cytokines in the intestine of LPS-induced inflammation with or without taVNS treatment. The relative expression levels of pro-inflammatory cytokines in response to taVNS treatment of LPS-induced endotoxemia were indicated. Fold change of groups was represented as a graph and calculated decreases rate in taVNS treated groups. (B) The morphological changes of the intestine were represented. Scale bar: 10 mm. (C) H&E staining showed the histological change of the intestine upon observation under a light microscope. Scale bar: 200 μm (upper panels) and 50 μm (lower panels). The villus height and crypto depth of the intestine were determined and represented by a graph. (D) Immunohistochemical staining with anti-MPO antibodies showed a significant decrease in neutrophils in LPS-induced inflammation by interventions as of taVNS. Scale bar: 50 μm. All taVNS groups were treated with electrical stimulation for 10 min. Data have been presented as the means and SD; * p < 0.05, ** p < 0.01, and *** p < 0.001, compared to the corresponding control.

Figure 6. Changes in inflammatory cytokines and chemokines in the plasma of LPS-induced endotoxemia with taVNS. (A) Most cytokines and chemokines were upregulated by LPS injection (LPS/Sham). Both taVNS groups (15 Hz and 25 Hz) attenuated the expression levels of cytokines and chemokines in serum. taVNS with 15 Hz was more significantly reduced than with 25 Hz. Clustering software generated a heatmap. The color depicts the levels of fold change. (B) Scatter plot analysis showed the expression changes of cytokines and chemokines for the LPS-induced inflammation verse sham (left), taVNS with 15 Hz on the LPS-induced inflammation versus the LPS-induced inflammation (middle), and taVNS with 25 Hz on the LPS-induced inflammation versus the LPS-induced inflammation (right). Upregulation is presented as red dots and downregulation as green dots. (C) The representative pixel density of pro- and anti-inflammatory-related cytokines, namely TNF-α, IL-6, IL-1β, TGF-β1, TLR4, and IL-10 in the serum of the sham, LPS applied groups and taVNS with 15 Hz or 25 Hz on LPS-induced groups. Data have been presented as the means and SD; * p < 0.05, ** p < 0.01, and *** p < 0.001 to determine the significance…